INTRODUCTION

Bonding is a significant procedure in fixed orthodontic treatment. Bonding errors can cause undesirable tooth positions and may increase total treatment time. Orthodontic attachments are bonded directly on the enamel surface with a semiflowable composite resin in the conventional direct bonding technique. Direct bonding is prone to misplacement, is even more difficult to use for lingual technique, and requires long chair time. Silverman and Cohen developed the indirect bonding technique to overcome these disadvantages.¹ Brackets are placed on plaster or digital dental models and are carried into the patient’s mouth with a “bonding tray” in the indirect bonding technique. Working on dental models in the laboratory and, more recently, using software programs, enabled detailed analysis of the bracket positions; therefore, the indirect technique significantly decreased bracket positioning errors.^1–3 However, there are disadvantages such as technique sensitivity, additional laboratory or digital processing, and the risk of adhesive leakage into the gingival embrasures.⁴

Bonding materials designed explicitly for indirect bonding techniques were required to achieve improved precision and bonding strength. Adhesive resins used in direct bonding are desired to be on the stiff side of viscosity to hold the bracket in the oriented position until it is cured. At the same time, indirect bonding adhesives tend to be less viscous to form a thin coating between the bracket base and enamel surface. Self-curing bonding resins are often used with nontransparent indirect bonding trays.

Monomers are an essential element in adhesive systems and composite resins and are key components that provide adhesion.⁵ Cross-linked polymers show better mechanical strength and are important for reinforcing composite resin⁵. Bisphenol A-glycidyl dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), and hydroxyethyl methacrylate (HEMA) are crosslinking dimethacrylates commonly used in the content of composite resins used in dentistry.

Studies have shown that deterioration products and monomers are released from adhesive systems.^6,7 It is essential to determine the monomers that directly affect the clinical success of adhesive systems and the amounts that can cause adverse biological effects.⁸ The DNA inhibition test performed by Heil et al.⁹ detected DNA damage due to BisGMA in some samples. It has been reported stated that HEMA and TEGDMA monomers can cause chromosomal aberrations at high concentrations.¹⁰ It is established that BisGMA is metabolized into Bisphenol A by hydrolytic and enzymatic degradation.¹¹

There is a need to study the monomer release of modern indirect bracket bonding systems. Therefore, the aim of this study was to determine the amounts of monomer released from indirect bonding adhesives using “ultra-high-performance liquid chromatography” (UHPLC), compare these amounts against a direct bonding adhesive, and comment on the potential harmful biological effects of these materials.

MATERIALS AND METHODS

Sample Preparation

Five-hundred stainless steel brackets (Gemini TM Series Low Profile, 3M Unitek, Monrovia, CA, USA) and bovine incisors were used. Roots of the teeth were cut from the cementoenamel junction. Teeth were cleaned with nonfluoridated pumice and washed under running tap water to eliminate pumice residue after removing the soft-tissue remnants. The bovine incisor teeth were disinfected by immersing them in a 5% sodium hypochlorite solution for 24 hours and then stored in distilled water until use.

Enamel preparation and bonding procedures were performed according to the manufacturer’s instructions. The bonded brackets were stored for 5 minutes at 37°C and 50% relative humidity for adequate polymerization. Each group had 100 bonded brackets. Group I and II were polymerized using LED (Elipar S10, 3M ESPE, St. Paul, MN, USA) with an output of 1200 mW/cm², while other groups had chemical-cure resins.

The brackets were randomly divided into five groups according to the adhesive systems:

Light Cure Adhesives: TXT (Transbond XT, 3M Unitek, Monrovia, CA, USA), SLV (Transbond Supreme LV, 3M Unitek).

Chemical Cure Adhesives: SRS (Sondhi Rapid-Set, 3M Unitek), IDB (Transbond IDB Pre-Mix, 3M Unitek), CIQ, (Custom I.Q., Reliance Orthodontic Products, Itasca, IL, USA).

Material properties are displayed in Table 1.

Table 1. Chemical Composition of Adhesive Materials^a

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All specimens were immersed in sterile glass tubes containing 15 mL of absolute alcohol (99% v/v) to induce accelerated aging after bonding. Alcohol was chosen for its known action to cause plasticizing and accelerate degradation.¹² The solution was shaken for 10 seconds twice a day during the immersion period.

An ultra-high-performance liquid chromatography (UHPLC, Dionex Ultimate 3000 RS, Sunnyvale, CA, USA) device was used to determine the amounts of released UDMA, TEGDMA, BisGMA, and HEMA monomers in the alcohol solutions. Liquid samples of 0.5 mL from each solution were removed on days 1, 7, 21, and 35. Liquid samples were filtered through 0.22 μm nylon filter paper, and the filtered liquid was injected into the opaque glass vials. The UHPLC device was equipped with a high-performance liquid chromatography (HPLC) gradient pump (Waters 600E, Milford, Mass), an injector (Rheo-dyne 7725i, Cotati, CA, USA) with a 20 μL sample loop, a reversed-phase Acquity UPLC BEH C18 analytical column (Waters, Milford, MA, USA) (150 mm × 2.1 mm ID, 1.7 μm), and a tunable absorbance detector (Waters 486). The analysis was conducted under the following conditions: acetonitrile/water (80:20 v/v) mobile phase; isocratic elution mode; 20 μL minimum flow rate; and detection at 254 nm.

The column was calibrated with known concentrations of BisGMA, TEGDMA, UDMA, and HEMA (Sigma-Aldrich, St Louis, MO, USA) in ethanol. The linear fittings of the calibration curves were used to calculate the concentration of monomers in the alcohol solution based on the area of chromatographic peaks at the corresponding retention time. The UHPLC assays were duplicated each time, and results were averaged.

With each adhesive used in the study, a tooth with an additional bracket was embedded in the acrylic block. The tooth was cut vertically through the middle of the bracket with a sectioning device (Buehler, Lake Bluff, IL, USA), and images were acquired by scanning electron microscope (SEM) (Quanta FEG 250, FEI Inc., USA). In addition, the amount and shape of the adhesive between the tooth surface and the bracket base was evaluated with images obtained at 60-, 100-, 500-, 1000-, 2000-, and 4000 times magnification.

RESULTS

Detailed statistical analyses are presented in the tables. The release values shown in the graphs and tables represent accumulated levels of monomers at each timepoint. Analysis of monomers is elaborated separately below to examine each monomer’s behavior in detail.

UDMA

The highest UDMA release was observed in the CIQ group, and the least was observed in the SLV group. TXT group release values were significantly lower (P < .01) than the IDB group. When UDMA release values were considered in all groups, the values on the first and seventh days were significantly lower (P < .01) than those on the twenty-first and thirty-fifth days (Figure 1a, Table 2).

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Table 2. Means and Standard Deviations (in ppm) of the UDMA Readings in Each Group Evaluated on Days 1, 7, 21, and 35^a,b

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TEGDMA

When TEGDMA release values were considered at all times, the values of TXT and SLV groups were significantly lower than those of SRS and IDB groups (P < .01). There was no statistically significant difference between TXT and SLV or between SRS and IDB groups. The highest TEGDMA release was observed in the IDB group, and the lowest TEGDMA release was observed in the TXT group. When the TEGDMA release values of all groups were considered, no significant difference was observed among the periods (Figure 1b, Table 3).

Table 3. Means and Standard Deviations (in ppm) of TEGDMA Readings in Each Group Evaluated on Days 1, 7, 21, and 35^a,b

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BisGMA

When the values of BisGMA release were considered, there was a statistically significant difference among the groups (P < .01). The least release occurred in the SLV group, and the most was in the CIQ group. In comparing the periods, the oscillation values on the first day were statistically lower than those on the 35th day (P < .01) (Figure 1c, Table 4).

Table 4. Means and Standard Deviations (in ppm) of the BisGMA Readings in Each Group Evaluated on Days 1, 7, 21, and 35^a,b

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HEMA

Considering the HEMA release values at all time periods, the lowest value was in the SLV group and the highest was in the SRS group. There was no difference among the SLV, TXT, and CIQ groups. There was also no difference between the CIQ and IDB groups. Comparing the periods, the release values on the first day were significantly lower than those on the thirty-fifth day (P < .01). There was no significant difference between the SLV and TXT groups. A statistically significant difference was observed among all other groups (P < .01) (Figure 1d, Table 5).

Table 5. Means and Standard Deviations (in ppm) of the HEMA Readings in Each Group Evaluated on Days 1, 7, 21, and 35^a,b

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The adhesive thicknesses between the tooth surface and the bracket base were measured in the following amounts: TXT (248.7 μm, 137.1 μm; Figure 2), SLV (206.7 μm, 94.45 μm; Figure 3), SRS (155.0 μm, 24.91 μm; Figure 4), IDB (187.8 μm, 64.76 μm; Figure 5), and CIQ (188.1 μm, 34.20 μm; Figure 6).

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For adhesive thicknesses between the tooth surface and the bracket base, the highest value was obtained with Transbond XT adhesive. In contrast, the lowest value was observed in the Sondhi Rapid-Set adhesive. The amount of adhesive protruding from the bracket edge was visually observed, at least in the Sondhi Rapid-Set adhesive. The most overflowing adhesive was observed in Custom I.Q.

DISCUSSION

Studies have shown that degradation products and residual monomers are released from adhesive systems.^6,7,13 As has been reported, the amount of residual monomers is associated with conditions such as: material composition, type and rates of inhibitors and initiators in its content, intensity and wavelength of the light produced by the light device used, as well as exposure time to light, ambient temperature, and oxygen in the environment.¹⁴

It was reported that there is a high amount of residual monomer release in the first minutes of the resin material polymerization, which gradually decreases with time.¹⁵ Researchers have reported that monomer release continued until the 30th day.⁶ Eliades et al.,¹⁶ Sunitha et al.,¹⁷ and Purushothaman et al.¹⁸ examined residual monomer release from orthodontic adhesives and determined the measurement periods as first, seventh, twenty-first, and thirty-fifth day.

Researchers used many solutions, such as distilled water, saline, ethanol, methanol, acetone, and artificial saliva. Moharamzadeh et al.¹⁹ observed many peaks in the chromatographic analyses caused by components in the formulation of artificial saliva, sodium and chloride ions in physiological saline, and organic and inorganic contents of the medium. As the increase in the number of peaks makes it difficult to define peaks consisting of monomers, such liquids were not used in this study. In this study, 99% ethanol was used to increase monomer release, accelerate the aging effect, and simulate variable oral cavity conditions.²⁰

It was stated that chromatographic methods are the most appropriate methods for monomer release analysis after polymerization from resin-based materials used in the dental area.²¹ The HPLC method is used to analyze larger molecular weight monomers such as UDMA, BisGMA, TEGDMA, and HEMA.^6,16,17,22 Researchers have examined the release of bisphenol-A (BPA), which is released by the hydrolytic and enzymatic degradation of BisGMA.^11,23 However, gas chromatography methods are needed for BPA analysis. Since the materials to be determined in the current study were liquid and their molecular weights are relatively low, HPLC analysis was considered a more suitable and reliable method.

HEMA release is seen in all groups because of high hydrophilicity, and the low molecular weight of HEMA is thought to have high mobility. Similarly, it was reported that the low molecular weight molecules such as HEMA and TEGDMA have a faster release rate than high molecular weight molecules such as BisGMA and UDMA.¹⁹ HEMA and UDMA values observed in the current study were consistent with this.

Many researchers investigated the correlation between residual monomers released from resin-based composites in culture media and cytotoxicity and found positive results. Also, cell death occurred in culture media around discs formed using these materials.^22,24,25

Wada et al.²⁶ analyzed the estrogenic effects of residual monomers released from composite discs kept at different concentrations and found that residual monomers had severe estrogenic effects. Olea et al.²⁷ also suggested that such monomers had a serious estrogenic effect using residual monomer analysis and recommended that such materials used in pediatric patients should be reviewed. Cytotoxicity levels of monomers used in resin materials were high to low in BisGMA, UDMA, TEGDMA, and HEMA.²⁸ Each of the adhesive materials used in this study contained at least three of the BisGMA, UDMA, TEGDMA, and HEMA monomers, which have potential for cytotoxic and hormonal effects.

In general, adhesives with low molecular weight monomers and less filler should form thinner layers due to their lower viscosity. This difference can be attributed to higher viscosities of densely filled composites, which require increased compressive forces to cause the material to thin. Additionally, thicker adhesives on the bracket base will have more surface area and be more affected by intraoral physical and chemical factors. In addition, adhesive thickness between the tooth surface and the bracket was evaluated in the current study, considering that monomer release would increase due to the surface area being significantly affected.

The thickness of adhesive layers in clinical conditions is estimated to vary between 100 and 250 μm, depending on the morphology and design of the bracket base.²⁹ Adhesive thicknesses in the current study were between these values. Differences in the appearance of residual adhesives are also attributed to monomers, fillers, and their viscosity in the adhesives.

CONCLUSIONS

• Monomer release in adhesives is inevitable. Therefore, clinicians should be aware of potential risks of orthodontic adhesives.

• As findings in this study were obtained in in vitro conditions for 35 days, further studies are needed to evaluate the amount of monomers released into the tooth and surrounding tissues under constantly changing conditions in the oral environment.

• Clinicians should remember that orthodontic adhesives contain an endocrine disrupter, allergenic molecules, and cytotoxic or genotoxic compounds. Manufacturers should ensure that they identify those substances in their products that can cause allergic or undesirable side effects for patients and dentists.

• Undoubtedly, the search for ideal adhesive materials will continue in orthodontics.